Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) produce profound motor and cognitive impairment associated with aggregation of ?-synuclein (?Syn). In these diseases, ?Syn undergoes complex post-translational modifications and fragmentation. In particular, many reports show increasing levels of C- terminally truncated (?C) ?Syn in brains of familial PD ?Syn mutation carriers and as a function of disease severity. Notably, truncated ?C-?Syn can induce soluble full-length ?Syn to co-assemble and thus accelerate, or even initiate, its aggregation. Limited research has focused on the proteases mediating ?C-?Syn truncation, their potential activation pathways, and the specific forms of the ?Syn substrate. Known caspase and calpain recognition sites occur in the ?Syn C-terminus, and inhibiting these proteases lowers ?C-?Syn and subsequent ?Syn aggregation. Moreover, the PI's recent studies of ?Syn in rodents exposed to paraquat, a herbicide linked to sporadic PD risk, showed elevated ?C-?Syn due to activation of calpain-1. However, these and other studies examining ?Syn in neurons or brain tissue have used denaturing conditions that preclude discriminating between monomers and the physiological ?Syn tetramer/multimers that our and other labs recently discovered. Using an intact-cell crosslinking assay that preserves the lysis-sensitive tetramers/multimers, we have shown that all fPD missense mutants shift normal tetramers to monomers. Further, we reported that mutating the N-terminal KTKEGV repeat motif, namely expressing E35K+E46K (?2K?) and E35K+E46K+E61K (?3K?) that mimic a doubling or tripling of the fPD E46K mutation, decreases the propensity of ?Syn to form normal tetramers/multimers. The resultant excess monomers associate with membranes, form ?Syn-rich vesicular inclusions, cause neurotoxicity, and could be subject to ?C truncation. Hence our central hypothesis is that excess ?Syn monomers arising under both familial and `sporadic' PD conditions provide the substrate for adverse proteolytic truncation. To pursue this hypothesis, we propose (Aim 1) to phenotype in detail our novel ?2K? and ?3K? tetramer-abrogating mice that develop neuronal aggregates in nigra and cortex containing ?C-?Syn and show progressive motor deficits with prominent tremor. As a distinct but complementary approach (Aim 2), we will apply paraquat to activate calpain-1 and to see if the resultant increase ?C-?Syn alters the multimer-to-monomer ratio by sequestering monomers into aggregates. Mechanistically, cholesterol-rich caveolar membranes regulate Ca2+ entry and calpain-1 activity, so we will ask if the calpain-mediated ?C truncation induced by paraquat or the fPD mutations is associated with changes of caveolar structure/structural proteins. In sum, we will generate new tools including novel tetramer-abrogating ?Syn tg mice and paraquat-treated neurons to identify ?Syn forms and examine proteases underlying the adverse ?C truncation that contributes to the progressive aggregation centrally implicated in PD and DLB.